Senile plaques, a major neuropathologic feature of Alzheimer's disease (AD), are composed largely of the amyloid peptide (Abeta) that is derived by processing of the amyloid precursor protein (APP). The two major forms of amyloid are 40 and 42 amino acid long. While Abeta(1-40) is more abundant, Abeta(1-40) aggregates more readily and comprises the majority of the amyloid in SPs. Mutations in APP that are associated with familial AD (FAD) alter the Abeta production by increasing the total amount of Abeta generated, or by causing a relative increase in the amount of Abeta(1-40) produced. Thus, perturbations in APP processing and Abeta production may play an important role in the development of AD. Therefore, the goals of Project 1 are to continue delineation of APP processing pathways in human neuronal model system (NT2N cells). We recently found that retention of APP in the endoplasmic reticulum/intermediate compartment (ER/IC) blocked Abeta secretion, but intracellular Abeta was still produced. Quantitative ELISA showed that ER/IC-associated Abeta is composed exclusively of Abeta(1-42). This intra-neuronal Abeta(1-42) accumulates over a period of weeks in NT2N while becoming less soluble. These observations have implications for understanding AD pathogenesis, including possible links to the presenilins (i.e., PS1 and PS2) which are membrane proteins largely localized to the ER/IC that cause AD when they are mutated in FAD pedigrees. Interestingly, FAD-associated forms of mutant the presenilins increased the levels of Abeta(1-42). Long-term accumulation of aggregated intra-neuronal Abeta(1-42) might be neurotoxic and ultimately lead to the formation of SPs following neuronal death. We propose to extend our studies on this novel pathway, to examine other subcellular compartments where Abeta is produced, and to study the effects of the presenilins on Abeta production through the following Specific Aims: 1) to continue our recent studies on the ER/IC pathway by which intracellular Abeta(1-42) is produced; 2) to study potential interactions between the presenilins and APP, and examine how expression of wild type or mutant PS1 and PS affects intracellular Abeta production; 3) to examine the role of the endocytic pathway in the production of intracellular and secreted Abeta(1-40) and Abeta(1-42); and 4) to study how wild-type and mutant forms of APP751 AND 770 are processed by NT2N neurons.